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• W2779759291 abstract "We recently developed a therapeutic biopolymer composed of an elastin-like polypeptide (ELP) fused to vascular endothelial growth factor (VEGF) and showed long-term renoprotective effects in experimental renovascular disease after a single intra-renal administration. Here, we sought to determine the specificity, safety, efficacy, and mechanisms of renoprotection of ELP-VEGF after systemic therapy in renovascular disease. We tested whether kidney selectivity of the ELP carrier would reduce off-target binding of VEGF in other organs. In vivo bio-distribution after systemic administration of ELP-VEGF in swine was determined in kidneys, liver, spleen, and heart. Stenotic-kidney renal blood flow and glomerular filtration rate were quantified in vivo using multi-detector computed tomography (CT) after six weeks of renovascular disease, then treated with a single intravenous dose of ELP-VEGF or placebo and observed for four weeks. CT studies were then repeated and the pigs euthanized. Ex vivo studies quantified renal microvascular density (micro-CT) and fibrosis. Kidneys, liver, spleen, and heart were excised to quantify the expression of angiogenic mediators and markers of progenitor cells. ELP-VEGF accumulated predominantly in the kidney and stimulated renal blood flow, glomerular filtration rate, improved cortical microvascular density, and renal fibrosis, and was accompanied by enhanced renal expression of VEGF, downstream mediators of VEGF signaling, and markers of progenitor cells compared to placebo. Expression of angiogenic factors in liver, spleen, and heart were not different compared to placebo-control. Thus, ELP efficiently directs VEGF to the kidney after systemic administration and induces long-term renoprotection without off-target effects, supporting the feasibility and safety of renal therapeutic angiogenesis via systemic administration of a novel kidney-specific bioengineered compound. We recently developed a therapeutic biopolymer composed of an elastin-like polypeptide (ELP) fused to vascular endothelial growth factor (VEGF) and showed long-term renoprotective effects in experimental renovascular disease after a single intra-renal administration. Here, we sought to determine the specificity, safety, efficacy, and mechanisms of renoprotection of ELP-VEGF after systemic therapy in renovascular disease. We tested whether kidney selectivity of the ELP carrier would reduce off-target binding of VEGF in other organs. In vivo bio-distribution after systemic administration of ELP-VEGF in swine was determined in kidneys, liver, spleen, and heart. Stenotic-kidney renal blood flow and glomerular filtration rate were quantified in vivo using multi-detector computed tomography (CT) after six weeks of renovascular disease, then treated with a single intravenous dose of ELP-VEGF or placebo and observed for four weeks. CT studies were then repeated and the pigs euthanized. Ex vivo studies quantified renal microvascular density (micro-CT) and fibrosis. Kidneys, liver, spleen, and heart were excised to quantify the expression of angiogenic mediators and markers of progenitor cells. ELP-VEGF accumulated predominantly in the kidney and stimulated renal blood flow, glomerular filtration rate, improved cortical microvascular density, and renal fibrosis, and was accompanied by enhanced renal expression of VEGF, downstream mediators of VEGF signaling, and markers of progenitor cells compared to placebo. Expression of angiogenic factors in liver, spleen, and heart were not different compared to placebo-control. Thus, ELP efficiently directs VEGF to the kidney after systemic administration and induces long-term renoprotection without off-target effects, supporting the feasibility and safety of renal therapeutic angiogenesis via systemic administration of a novel kidney-specific bioengineered compound. The outcomes of renovascular disease (RVD) are still poor, and there is a noticeable lack of consensus regarding the best therapeutic strategy for these patients, which adds a burden of uncertainty to the treatment selection and course. Regardless of the chosen therapy (medical, interventional, or combined therapy), patients with RVD improve in ∼30% of the cases.1Choi S.S. Atherosclerotic renal artery stenosis and revascularization.Exp Rev Cardiovasc Ther. 2014; 12: 1419-1425Crossref PubMed Scopus (5) Google Scholar, 2Yu M.S. Folt D.A. Drummond C.A. et al.Endovascular versus medical therapy for atherosclerotic renovascular disease.Curr Atheroscler Rep. 2014; 16: 459Crossref PubMed Scopus (6) Google Scholar Furthermore, the results of the CORAL study support the notion that pharmacologic or interventional (e.g., renal angioplasty to resolve the obstruction) strategies do not show significant differences in renal recovery to support one treatment over the other,3Cooper C.J. Murphy T.P. Cutlip D.E. et al.Stenting and medical therapy for atherosclerotic renal-artery stenosis.N Engl J Med. 2014; 370: 13-22Crossref PubMed Scopus (648) Google Scholar although secondary evaluations of the ASTRAL study suggest that interventional strategies may still be beneficial in selected populations.4Ritchie J. Green D. Chrysochou C. et al.High-risk clinical presentations in atherosclerotic renovascular disease: prognosis and response to renal artery revascularization.Am J Kidney Dis. 2014; 63: 186-197Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar These controversies feed a pressing need for novel and more effective therapeutic strategies for the growing population of patients suffering from RVD that are at higher cardiovascular risk and at risk of the development of chronic kidney disease (CKD). It is possible that the vascular obstruction in RVD may be a major instigator of renal injury, and it may also exacerbate pre-existing renal damage.5Textor S.C. Lerman L.O. Reality and renovascular disease: when does renal artery stenosis warrant revascularization?.Am J Kidney Dis. 2014; 63: 175-177Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 6Textor S.C. Misra S. Oderich G.S. Percutaneous revascularization for ischemic nephropathy: the past, present, and future.Kidney Int. 2013; 83: 28-40Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar However, the dynamic and progressive nature of RVD may be a driving force for evolving renal injury distal to the vascular obstruction and may determine the chances of renal recovery after therapeutic interventions. Thus, it is possible that the poor recovery in RVD results from a combination of doing too little or acting too late with the possibility of neglecting the stenotic renal parenchyma. Renal microvascular (MV) dysfunction, remodeling, and even loss are hallmarks of CKD irrespective of the etiology.7Mack M. Yanagita M. Origin of myofibroblasts and cellular events triggering fibrosis.Kidney Int. 2015; 87: 297-307Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, 8Xavier S. Vasko R. Matsumoto K. et al.Curtailing endothelial TGF-beta signaling is sufficient to reduce endothelial-mesenchymal transition and fibrosis in CKD.J Am Soc Nephrol. 2015; 26: 817-829Crossref PubMed Scopus (110) Google Scholar We have shown that damage of the small vessels in the kidney correlates with a significant deterioration of renal hemodynamics, filtration, and tubular function in a swine model of chronic RVD.9Chade A.R. Rodriguez-Porcel M. Grande J.P. et al.Distinct renal injury in early atherosclerosis and renovascular disease.Circulation. 2002; 106: 1165-1171Crossref PubMed Scopus (223) Google Scholar, 10Chade A.R. Zhu X. Mushin O.P. et al.Simvastatin promotes angiogenesis and prevents microvascular remodeling in chronic renal ischemia.FASEB J. 2006; 20: 1706-1708Crossref PubMed Scopus (116) Google Scholar, 11Iliescu R. Fernandez S.R. Kelsen S. et al.Role of renal microcirculation in experimental renovascular disease.Nephrol Dial Transplant. 2010; 25: 1079-1087Crossref PubMed Scopus (102) Google Scholar We also demonstrated that MV disease in the stenotic kidney is associated with blunted renal angiogenesis, which is driven by a progressive decrease in renal vascular endothelial growth factor (VEGF).10Chade A.R. Zhu X. Mushin O.P. et al.Simvastatin promotes angiogenesis and prevents microvascular remodeling in chronic renal ischemia.FASEB J. 2006; 20: 1706-1708Crossref PubMed Scopus (116) Google Scholar, 12Zhu X.Y. Chade A.R. Rodriguez-Porcel M. et al.Cortical microvascular remodeling in the stenotic kidney: role of increased oxidative stress.Arterioscler Thromb Vasc Biol. 2004; 24: 1854-1859Crossref PubMed Scopus (133) Google Scholar The pivotal role of this proangiogenic cytokine in the kidney is supported by proof of concept studies showing that intrarenal replenishment of VEGF ameliorated renal MV rarefaction and attenuated renal dysfunction and damage.11Iliescu R. Fernandez S.R. Kelsen S. et al.Role of renal microcirculation in experimental renovascular disease.Nephrol Dial Transplant. 2010; 25: 1079-1087Crossref PubMed Scopus (102) Google Scholar, 13Chade A.R. Kelsen S. Renal microvascular disease determines the responses to revascularization in experimental renovascular disease.Circ Cardiovasc Interv. 2010; 3: 376-383Crossref PubMed Scopus (57) Google Scholar, 14Chade A.R. Kelsen S. Reversal of renal dysfunction by targeted administration of VEGF into the stenotic kidney: a novel potential therapeutic approach.Am J Physiol Renal Physiol. 2012; 302: F1342-F1350Crossref PubMed Scopus (59) Google Scholar We recently extended and refined renal VEGF therapy. We developed a novel fusion of a bioengineered protein for drug delivery with VEGF121. We used elastin-like polypeptides (ELPs), which are genetically encoded drug-delivery vectors with long plasma half-life, low immunogenicity, and adaptability to be fused to nearly any therapeutic. Furthermore, ELPs naturally accumulate in kidney.15Bidwell 3rd, G.L. Mahdi F. Shao Q. et al.A kidney-selective biopolymer for targeted drug delivery.Am J Physiol Renal Physiol. 2017; 312: F54-F64Crossref PubMed Scopus (50) Google Scholar, 16Bidwell 3rd, G.L. Perkins E. Hughes J. et al.Thermally targeted delivery of a c-Myc inhibitory polypeptide inhibits tumor progression and extends survival in a rat glioma model.PloS One. 2013; 8: e55104Crossref PubMed Scopus (84) Google Scholar, 17Bidwell 3rd, G.L. Perkins E. Raucher D. A thermally targeted c-Myc inhibitory polypeptide inhibits breast tumor growth.Cancer Lett. 2012; 319: 136-143Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 18Chade A.R. Tullos N.A. Harvey et al.Renal Therapeutic Angiogenesis Using a Bioengineered Polymer-Stabilized Vascular Endothelial Growth Factor Construct.J Am Soc Nephrol. 2016; 27: 1741-1752Crossref PubMed Scopus (45) Google Scholar The ELP-VEGF construct displayed a prolonged circulation and tissue residence time, improved stenotic kidney targeting, and long-term efficacy of VEGF therapy in the RVD model (compared with unconjugated VEGF) after a single intrarenal administration.18Chade A.R. Tullos N.A. Harvey et al.Renal Therapeutic Angiogenesis Using a Bioengineered Polymer-Stabilized Vascular Endothelial Growth Factor Construct.J Am Soc Nephrol. 2016; 27: 1741-1752Crossref PubMed Scopus (45) Google Scholar However, whether systemic administration of ELP-VEGF targets and protects the kidney is unknown and is important to determine from a clinical/translational perspective. Thus, we first seek to establish the renal specificity of the ELP-VEGF construct after systemic administration, as we also aim to determine the safety and efficacy in the RVD model through this route. We hypothesize that the fusing of VEGF to the ELP biopolymer carrier will lead to renal tissue specificity and kidney accumulation without decreasing therapeutic efficacy even after systemic administration. Finally, we intend to determine potential off-target effects (a concern from a clinical/translational perspective) and underlying mechanisms of long-term renoprotection after systemic ELP-VEGF therapy. Labeling was performed on primary amine residues, including the protein’s N-terminus, its 1 lysine residue near the N-terminus, and its 8 lysine residues on the surface of VEGF121 as highlighted in Supplementary Figure S1. Labeling did not alter VEGF potency. For more details, see Supplementary File. The stability of the ELP-VEGF biopolymer was determined in vitro, as described in the Supplementary File. As shown in Supplementary Figure S2A, ELP-VEGF was present as a single band migrating at 74 kDa, and the free rhodamine label migrated below the 10-kDa marker. When incubated in phosphate-buffered saline, very little ELP-VEGF degradation was observed for the first 24 hours (quantified in Supplementary Figure S2B). Degradation of the protein began between 24 and 48 hours of incubation and proceeded to nearly complete loss of the full-length band after 4 days. A similar slow degradation was observed when ELP-VEGF was incubated in plasma. The kinetics of the degradation in plasma were different from the PBS incubation, beginning more quickly. However, after 5 days in plasma, more full-length protein remained intact than in the phosphate-buffered saline incubation, possibly reflecting a stabilizing decoy effect of other plasma proteins occupying proteases. Detection of free dye by using trichloroacetic acid to precipitate the protein component of each sample mirrored the gel electrophoresis data in the phosphate-buffered saline incubation, with free dye slowly being released over a period between 20 and 96 hours. However, the amount of free dye peaked at only ∼25%, indicating that most of the dye was still bound to a protein component and consistent with the presence of a band of ∼10 kDa in Supplementary Figure S2A. After incubation in plasma, almost no free dye was detectable, although it is possible that some free dye bound to albumin or other plasma proteins and was thus precipitated by trichloroacetic acid. These analyses reveal that ELP-VEGF does degrade under physiologic conditions, but the rate of degradation is quite slow relative to the rate of clearance from the body observed here and in other studies.18Chade A.R. Tullos N.A. Harvey et al.Renal Therapeutic Angiogenesis Using a Bioengineered Polymer-Stabilized Vascular Endothelial Growth Factor Construct.J Am Soc Nephrol. 2016; 27: 1741-1752Crossref PubMed Scopus (45) Google Scholar, 19George E.M. Liu H. Robinson G.G. et al.Growth factor purification and delivery systems (PADS) for therapeutic angiogenesis.Vasc Cell. 2015; 7: 1Crossref PubMed Scopus (27) Google Scholar To determine the pharmacokinetics and biodistribution of ELP-VEGF, the protein was fluorescently labeled, and plasma levels and organ biodistribution were determined 4 hours after a single i.v. administration (ear vein catheter) in the swine. The fluorescently labeled protein was administered i.v. in swine at a bolus dose of 1 mg/kg, plasma was sampled intermittently, and organ fluorescence was determined at killing 4 hours after injection. Whole-organ imaging revealed that ELP-VEGF predominantly accumulated in the kidney (Figure 1a). When the kidneys were cut in cross section, fluorescence imaging revealed ELP-VEGF localized at high levels in the renal cortex, with additional focal medullary localization in what are likely the large vascular branches. Retention of ELP-VEGF in the kidney was 3.2-fold higher than in the next most abundant organ, the liver. Additionally, ELP-VEGF levels in the kidney were 14.7-fold higher than in the lung, and the protein was undetectable in the heart and spleen at this dose (Figure 1b). Direct measurement of plasma fluorescence revealed a biphasic clearance of ELP-VEGF from the blood. A rapid distribution phase was evident within 30 minutes of injection, followed by a very slow clearance phase (Figure 1c). The slow clearance of ELP-VEGF is consistent with our observations of this protein after intrarenal administration, where we observed a half-life of ∼13.5 hours.18Chade A.R. Tullos N.A. Harvey et al.Renal Therapeutic Angiogenesis Using a Bioengineered Polymer-Stabilized Vascular Endothelial Growth Factor Construct.J Am Soc Nephrol. 2016; 27: 1741-1752Crossref PubMed Scopus (45) Google Scholar However, the short duration of this experiment did not provide enough clearance time to achieve an accurate fit of the terminal half-life following i.v. administration. Overall, these results demonstrate that ELP-VEGF is sufficiently stable under physiologic conditions, clears slowly, and most of the injected protein is retained in the kidney even after systemic injection using an ear vein route, suggesting that ELP-VEGF is renal selective and that systemic administration is a viable route for delivery of ELP-VEGF for renal therapy in RVD. In addition to determining the pharmacokinetics and biodistribution of ELP-VEGF, blood from the pigs before injection and 4 hours after injection was assessed to determine whether ELP-VEGF caused any acute effects. Quantification of blood urea nitrogen (BUN), creatinine, BUN/creatinine ratio, alanine aminotransferase, aspartate aminotransferase, γ-glutamyl transferase, and lactate dehydrogenase showed that a single administration of ELP-VEGF did not induce any acute changes in parameters of renal or liver function, suggesting a lack of acute toxicity and underscoring the safety of the construct (Table 1).Table 1Parameters of renal and liver function (mean ± SEM) at time 0 and 4 hours after the administration of ELP-VEGF i.v. injection (single dose, 1 mg/kg) for pharmacokinetics and biodistribution studiesParameterPlasma at baselinePlasma at 4 hrRangeP valueBUN (mg/dl)5.3 ± 0.65.33 ± 0.38–240.9Creatinine (mg/dl)1.2 ± 0.11.3 ± 0.11–30.69BUN/creatinine ratio4.56 ± 0.54.39 ± 0.60.85ALT (U/l)38.0 ± 7.734.5 ± 4.631–580.73AST (U/l)29.3 ± 6.734.7 ± 9.932–840.68GGT (U/l)42.3 ± 13.332.0 ± 10.810–600.57LDH (U/l)484.3 ± 159.8513.0 ± 121.5380–6300.89ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; GGT, γ-glutamyl transferase; LDH, lactate dehydrogenase. Open table in a new tab ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; GGT, γ-glutamyl transferase; LDH, lactate dehydrogenase. We then sought to determine whether a single i.v. dose of ELP-VEGF was safe and efficacious for improving renal function and decreasing renal injury, and whether off-target effects were observed. Body weight was similar among controls, RVD, and RVD+ELP-VEGF pigs 6 weeks after sham or induction of RVD and before treatment (Table 2). Pigs randomized to RVD and RVD+ELP-VEGF groups had elevated pretreatment blood pressure relative to non-RVD controls, but the RVD+placebo (saline) and RVD+ELP-VEGF pretreatment blood pressures were not significantly different from one another (Table 2). All animals were monitored during ELP-VEGF administration to determine the potential impact on heart rate and blood pressure, which were unchanged during injection. However, RVD+ELP-VEGF treated pigs showed a nonsignificant transient and asymptomatic decrease in blood pressure during the 24 to 48 hours following i.v. administration of the construct (from 142.5 ± 10.3 to 126.2 ± 9.7 mm Hg, P = not significant, Supplementary Figure S3) that then returned to hypertension pretreatment values. Hypertension was not significantly different between RVD and RVD+ELP-VEGF pigs at 10 weeks (Table 2, Supplementary Figure S3).Table 2Body weight, degree of stenosis, mean arterial pressure, renal vascular resistance, and renal cortical and medullary volumes (mean ± SEM) in normal, RVD, and RVD pigs before treatment (6 weeks after induction of RVD or sham operation) and 4 weeks after saline or ELP-VEGF treatment (10 weeks) (N = 6–7 per group)ParameterNormalRVDRVD+ELP-VEGFPretreatment values (6 wk)Body weight (kg)43.2 ± 3.845.1 ± 2.146.6 ± 2.8Degree of stenosis (%)0.0 ± 0.075.1 ± 7.2aP < 0.05 versus normal.73.7 ± 9.3aP < 0.05 versus normal.MAP (mm Hg)96.0 ± 2.4139.1 ± 9.3aP < 0.05 versus normal.142.5 ± 10.3aP < 0.05 versus normal.RVR (mm Hg/ml per min)0.21 ± 0.020.58 ± 0.04aP < 0.05 versus normal.0.64 ± 0.05aP < 0.05 versus normal.Cortical volume (ml)117.8 ± 7.060.4 ± 6.3aP < 0.05 versus normal.61.8 ± 7.2aP < 0.05 versus normal.Medullary volume (ml)34.0 ± 5.317.4 ± 2.0aP < 0.05 versus normal.18.1 ± 1.9aP < 0.05 versus normal.Posttreatment values (10 wk)Body weight (kg)51.3 ± 5.254.2 ±2.954.5 ± 2.8Degree of stenosis (%)0.0 ± 0.074.9 ± 6.5aP < 0.05 versus normal.75.2 ± 8.7aP < 0.05 versus normal.MAP (mm Hg)100.1 ± 1.9150.4 ± 10.4aP < 0.05 versus normal.142.4 ± 3.8aP < 0.05 versus normal.RVR (mm Hg/ml per min)0.19 ± 0.010.56 ± 0.07aP < 0.05 versus normal.0.47 ± 0.06aP < 0.05 versus normal.,bP < 0.05 versus RVD.Cortical volume (ml)96.3 ± 6.364.3 ± 8.7aP < 0.05 versus normal.64.2 ± 5.9aP < 0.05 versus normal.Medullary volume (ml)31.1 ± 6.518.4 ± 2.1aP < 0.05 versus normal.21.2 ± 7.0aP < 0.05 versus normal.ELP-VEGF, elastin-like polypeptides vascular endothelial growth factor; MAP, mean arterial pressure; RVD, renovascular disease; RVR, renal vascular resistance.a P < 0.05 versus normal.b P < 0.05 versus RVD. Open table in a new tab ELP-VEGF, elastin-like polypeptides vascular endothelial growth factor; MAP, mean arterial pressure; RVD, renovascular disease; RVR, renal vascular resistance. Computed tomography (CT)–derived stenotic kidney RBF, GFR, cortical and medullary perfusion were similarly decreased in all pigs with RVD after 6 weeks of observation, which correlated with a significant increase in renal vascular resistance (RVR) of the stenotic kidney (Table 2) and were accompanied by increased plasma creatinine (not shown). Blunted RBF, GFR (Figure 2), and regional perfusion (cortex: 3.3±0.5 ml/min/cc; medulla: 1.7±0.4 ml/min/cc) remained unchanged in RVD at 10 weeks (p=NS vs. 6 weeks) but RBF, GFR (Figure 2), and cortical perfusion (4.1±0.2 ml/min/cc, p<0.05 vs. 6 weeks) were stimulated after ELP-VEGF therapy whereas GFR showed a trend for a larger increase compared to pretreatment values at 6 weeks (Figure 2). Improvements in RVR (Table 2) and a plateau in plasma creatinine (which continued to increase in untreated RVD [Figure 2]) accompanied the improvements in renal function, suggesting slower or halted progression of renal dysfunction after ELP-VEGF therapy. The stenotic kidney showed a significant reduction in cortical and medullary MV density (quantified by micro-CT) accompanied by substantial MV remodeling compared with normal controls. Notably, systemically administered ELP-VEGF significantly improved cortical (but not medullary) MV density and remodeling of small and large microvessels (0–500 μm in diameter), which was evident throughout the renal cortical parenchyma (Figure 3a). This was accompanied by improved stenotic kidney media-to-lumen ratio, suggesting protection of the pre-existing microvasculature (Figure 3b). Plasma collected before treatment and four weeks after treatment in the RVD pigs was assessed with standard kidney and liver function assays to determine whether treatment caused any long-term effects. BUN was reduced at the 10-week time point relative to pretreatment values (Table 3). Creatinine levels were stable after treatment, consistent with the enzyme-linked immunosorbent assay data shown in Figure 2, and the BUN/creatinine ratio was significantly reduced by ELP-VEGF therapy. All measures of liver function were unchanged after ELP-VEGF treatment, and values were within normal ranges after treatment. Finally, no tumors were observed after ELP-VEGF therapy in any major organ.Table 3Kidney and liver parameters (mean ± SEM) after 6 weeks of RVD (before treatment) and then 4 weeks after administration of ELP-VEGF i.v. injectionParameterPlasma at 6 wkPlasma at 10 wkNormal rangeP valueBUN (mg/dl)8.3 ± 0.75.0 ± 0.58–240.03aP < 0.05, 10 versus 6 weeks (pretreatment).Creatinine (mg/dl)1.4 ± 0.11.5 ± 0.081–30.64BUN/creatinine ratio6. 1 ± 0.53.4 ± 0.10.04aP < 0.05, 10 versus 6 weeks (pretreatment).ALT (U/l)43.7 ± 6.550.7 ± 3.731–580.91AST (U/l)38.0 ± 2.232.3 ± 3.332–840.16GGT (U/l)32.3 ± 4.329.0 ± 2.310–600.70LDH (U/l)488.7 ± 19.6487.3 ± 63.9380–6300.91ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; ELP-VEGF, elastin-like polypeptides vascular endothelial growth factor; GGT, γ-glutamyl transferase; LDH, lactate dehydrogenase; RVD, renovascular disease.a P < 0.05, 10 versus 6 weeks (pretreatment). Open table in a new tab ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; ELP-VEGF, elastin-like polypeptides vascular endothelial growth factor; GGT, γ-glutamyl transferase; LDH, lactate dehydrogenase; RVD, renovascular disease. Protein extracts were made from the stenotic kidney harvested after killing at the 10-week time point and examined by Western blot to determine the effects of ELP-VEGF treatment on VEGF downstream signaling. A single systemic administration of ELP-VEGF improved the expression of proangiogenic hypoxia induced factor (HIF)-1α (upstream mediator of VEGF) and hepatocyte growth factor (HGF), VEGF and its receptor Flk-1, cMet (HGF was unchanged), and the expression of downstream mediators of VEGF signaling such as phosphorylated akt, phosphorylated extracellular signal–regulated kinase 1/2, and phospho-endothelial NO synthase (Figure 4a).Figure 4Systemic administration of elastin-like polypeptide vascular endothelial growth factor (ELP-VEGF) improved angiogenic signaling and stimulated progenitor cells in the stenotic kidney. (a) Representative renal protein expression (2 bands per group) and quantification of VEGF and its receptor Flk-1 (top), hypoxia-induced factor (HIF)-1α, hepatocyte growth factor (HGF), and HGF receptor cMet (middle), and total and phosphorylated akt, ERK1/2, and endothelial NOS (eNOS, bottom) in normal, renovascular disease (RVD), and RVD+ELP-VEGF stenotic kidneys. (b) Representative images of immunoreactivity against Oct-4 (×40), stromal-derived factor-1 (×20), and CD-34 (×20) and quantification in normal, RVD, and RVD+ELP-VEGF stenotic kidneys. *P < 0.05 versus Normal; †P < 0.05 versus RVD; #P > 0.1 versus RVD. RVD-ELP-VEGF IV, renovascular disease-elastin-like polypeptide-vascular endothelial growth factor IV; p-ERK, phosphorylated ERK. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Blocks of renal cortex were collected from the stenotic kidney after pigs were killed and fixed and stained for markers of progenitor cells. The improvements in VEGF signaling in these kidneys were also accompanied by enhanced renal immunoreactivity against CD-34, stromal-derived factor-1, and Oct-4 (mainly observed in the tubulointerstitial compartments and in MV proximity), suggesting potential stimulation, mobilization, and homing of systemic progenitor cells to the stenotic kidney and/or activation of resident cell progenitors in the stenotic kidney (Figure 4b). Concurrent with the improved renal function and MV remodeling, the stenotic kidney showed a decrease in the fraction of terminal deoxynucleotidyltransferase–mediated end-labeling positive cells and attenuated glomerulosclerosis and cortical tubulointerstitial fibrosis compared with untreated animals, suggesting a reduction in apoptosis and fibrosis (Figure 5b,c). Systemic administration may result in off-target accumulation and binding of the construct to tissues other than the stenotic kidney and stimulate vascular growth. Therefore, protein expression of angiogenic mediators was determined by Western blot in the contralateral kidney, liver, spleen, and heart of the untreated and treated RVD pigs. We observed that 4 weeks after 1 systemic administration of the ELP-VEGF construct, there were no changes in the expression of angiogenic mediators (except for a nonsignificant trend of Flk-1 in the contralateral kidney of the ELP-VEGF–treated animals), which suggests minimum off-target activity of the construct (Figure 6). The purpose of the current study was multifold. We first aimed to determine the feasibility and efficacy of targeting the kidney through systemic administration of ELP-VEGF therapy based on the higher preference of ELP for the kidney compared with other organs.18Chade A.R. Tullos N.A. Harvey et al.Renal Therapeutic Angiogenesis Using a Bioengineered Polymer-Stabilized Vascular Endothelial Growth Factor Construct.J Am Soc Nephrol. 2016; 27: 1741-1752Crossref PubMed Scopus (45) Google Scholar Determining a potential targeted renal therapy via a systemic administration could have implications for clinical application. We observed that a single administration of ELP-VEGF using a peripheral vessel was able to target the kidney and accumulate in the renal parenchyma, which was accompanied by improved renal function, improved angiogenic, reduced apoptotic signaling (which may precede renal fibrosis20Kitamura H. Shimizu A. Masuda Y. et al.Apoptosis in glomerular endothelial cells during the development of glomerulosclerosis in the remnant-kidney model.Exp Nephrol. 1998; 6: 328-336Crossref PubMed Scopus (68) Google Scholar), attenuated renal MV rarefaction, and reduced fibrosis, indicating that the effects of ELP-VEGF therapy in the stenotic kidney were not limited to vascular proliferation and repair. However, VEGF is a highly ubiquitous cytokine with autocrine, paracrine, and receptor-driven endocrine effects21Eichmann A. Simons M. VEGF" @default.
• W2779759291 created "2018-01-05" @default.
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• W2779759291 date "2018-04-01" @default.
• W2779759291 modified "2023-10-13" @default.
• W2779759291 title "Systemic biopolymer-delivered vascular endothelial growth factor promotes therapeutic angiogenesis in experimental renovascular disease" @default.
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• W2779759291 doi "https://doi.org/10.1016/j.kint.2017.09.029" @default.
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